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Overview and Sources of Reactive Oxygen Species (ROS) in the Reproductive

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Chapter 1
Overview and Sources of Reactive Oxygen
Species (ROS) in the Reproductive System

Gulfam Ahmad, Mazen Almasry, Amolak S. Dhillon, Muna M. Abuayyash,

Narasimhan Kothandaraman, and Zeynep Cakar

1.1  Introduction

Reactive oxygen species (ROS) are highly reactive molecules that are generated
from oxygen metabolism. They can be free radicals or non-radicals. Free radicals are
molecules that contain at least one unpaired valence electron at their outer shell,
making them highly reactive and short lived [1]. Among all the ROS, superoxide
anion (•O2−), hydrogen peroxide (H2O2) and hydroxyl radicals (•OH) are the most
known examples. Reactive nitrogen species (RNS), is the subclass of ROS that con-
tain nitrogen compound [2]. Both ROS and RNS, when present in physiological
amount, have important roles in normal cellular functions such as fighting against
infection, regulating different intercellular signaling pathways and facilitating
normal maturation and fertilization in reproductive systems [1, 3–7]. However, when

G. Ahmad (*)
College of Medicine, Prince Sattam Bin Abdulaziz University, Al Kharj, KSA, Saudi Arabia
Department of Physiology, University of Health Sciences, Lahore, Pakistan
e-mail: gulfam.uhs@gmail.com
M. Almasry
Alfaisal University, Riyadh, Saudi Arabia
A.S. Dhillon
University of British Columbia, Vancouver, BC, Canada
M.M. Abuayyash
Royal College of Surgeons in Ireland-Bahrain, Bahrain
N. Kothandaraman
American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, USA
Z. Cakar
Department of Histology and Embryology, Faculty of Medicine, Ankara University,
Ankara, Turkey

© Springer International Publishing AG 2017 1

A. Agarwal et al. (eds.), Oxidative Stress in Human Reproduction,
DOI 10.1007/978-3-319-48427-3_1
2 G. Ahmad et al.

ROS present in high concentration, overwhelming the antioxidant defense system,

oxidative stress results, and this may lead to cellular dysfunction via lipid peroxida-
tion, protein and DNA damages [8]. Due to such damaging effect on the cells, OS is
related to many pathological conditions including infertility [3, 9].
This chapter introduces concepts of free radicals, redox reactions, and then
focuses on ROS, RNS and their sources at cellular levels, particularly in both male
and female reproductive systems as well as covering a brief overview of oxidative
stress.

1.1.1  Free Radicals

Free radicals are molecules that contain at least one unpaired valence electron, mak-
ing them highly reactive and short-lived [1]. Among all the free radicals, hydroxyl
radical (•OH), and superoxide anion (•O2−) are the most known examples. They
derive from molecular oxygen under reducing conditions; however, because of their
reactivity, these free radicals can have deleterious effect on normal cellular func-
tions when present in high concentrations [1]. Other examples of free radicals are
hydroxyl radical (•OH), superoxide anion (•O2−), nitric oxide (•NO), lipid peroxyl
(•LOO−) and thiyl (•RS) [9].

1.1.1.1  Superoxide Anion (O2•−)

Univalent reduction of oxygen will result in the formation of superoxide anion

(O2•−) [1]. This reaction can happen in the mitochondrial electron transport chain
(ETC) without the involvement of any enzymatic activity by the action of oxidation-­
reduction reactive intermediates such semi-ubiquinone [10]. On the other hand,
enzymes such as nicotinamide adenine dinucleotide phosphate (NADPH) oxidases
and xanthine oxidase (XO) can also mediate this reaction. High amounts of super-
oxide anions are usually released from neutrophils and macrophages by the action
of different isoforms of NADPH oxidase. These superoxide anions can then be
converted into non-radical species, hydrogen peroxide (H2O2) and singlet oxygen
(1O2), enzymatically with oxidative enzymes such as superoxide dismutase (SOD)
or nonenzymatically [4].

e − + O2 → O2 •−
SOD
2O2 • +2H + → O2 + H 2 O2
−
1  Overview and Sources of Reactive Oxygen Species (ROS) in the Reproductive System 3

1.1.1.2  Hydroxyl Radical (•OH)

The interaction between hydrogen peroxide (H2O2) and reduced transition metals
such as copper and iron results in Fenton reaction, which leads to the formation of
hydroxyl radical (•OH). In addition, the exposure of water molecules to ionizing
radiation can also result in the production of hydroxyl radical [1, 11, 12].

Fe 2 + + H 2 O2 → Fe 3+ + OH − + •OH

1.1.2  Oxidation-Reduction (Redox) Reactions

Redox reactions or oxidation–reduction reactions are reactions that involve transfer

of electrons between two chemicals. The number of electrons accepted, lost or
shared is known as the oxidation number. When the oxidation number of any chemi-
cal increases, it is said that this chemical is oxidized and the process is known as
oxidation. In contrast, when the oxidation number is decreased, the chemical is
reduced and the process is known as reduction C [12, 13]. Iron rust is a good
example of a redox reaction. As shown below, iron (Fe) loses electrons to oxygen.
Hence, the oxidation number of iron increases while the oxidation number of oxygen
decreases. In other words, iron is oxidized while oxygen is reduced at the same
time. This is what happens when an iron nail rusts [13].

4 Fe 2 + + O2 → 4 Fe 3+ + 2O2 −

1.1.2.1  Haber-Weiss Reaction

Haber-Weiss reaction is another example of a redox reaction. In Haber-Weiss reaction,

superoxide anion interacts with hydrogen peroxide in the presence of a metal ion
(as a catalyst) to produce a hydroxyl radical [13]. The net reaction can be broken
down into two reactions. The first step involves the reduction of ferric to ferrous ion;
followed by the second step, the Fenton reaction. Along with the Fenton reaction,
these two reactions are important in generating the highly reactive hydroxyl radicals
[14, 15].

Reaction1 : Fe 3+ + O2 •− → Fe 2 + + O2

Reaction 2 : Fe 2 + + H 2 O2 → Fe 3+ + OH − + • OH

Net Reaction : O2 •− +H 2 O2 → • OH + OH − + O2

4 G. Ahmad et al.

Fig. 1.1  Generation of ROS via electron transport chain

1.1.2.2  Electron Transport Chain (ETC)

Electrons passage through a couple of specialized enzymes known as ETC in the mito-
chondria is important in cellular energy (adenosine triphosphate, ATP) production.
It involves a series of redox reactions. Electrons first enter the chain after the reduction
of NADH and FADH2, both of which act as electron carriers. Cytochrome oxidase, acts
as the final acceptor of these electrons, enhances the tetravalent reduction of oxygen
into water through a process known as oxidative phosphorylation [16]. During this
process, a small amounts of oxygen (2–5 %) are univalently reduced into free radicals
[17]. Ubisemiquinone, a part of ETC, is considered the main site of electron leak
leading to the generation of superoxide anions (Fig. 1.1) [4].

1.1.3  Reactive Oxygen Species (ROS)

The term reactive oxygen species (ROS) includes the reduced form of oxygen and
their reaction products with other molecules. Some but not all ROS are free radi-
cals [1]. Superoxide anion and hydroxyl radical are examples of radical ROS,
whereas hydrogen peroxide and hypochlorite ion are non-radical ROS (Table 1.1).
ROS usually have high reactivity due to the extra-unpaired electron at their outer
shells and their half-lives are in the order of milliseconds [10, 18, 19]. The hydroxyl
radical is the most damaging radical because it cannot be scavenged easily in our
bodies [1, 20].
Intracellular ROS can be produced by stimulation of tightly regulated NADPH
intracellular oxidases and peroxidases or as an alternate product by other enzymes
1  Overview and Sources of Reactive Oxygen Species (ROS) in the Reproductive System 5

Table 1.1  Radical and non-radical reactive oxygen species (ROS)

ROS
Free radicals Non-radicals
Hydroxyl radical (•OH) Hydrogen peroxide (H2O2)
Superoxide anion (•O2−) Singlet oxygen (1O2)
Lipid peroxyl (•LOO−) Ozone (O3)
Thiyl (•RS) Lipid peroxide (LOOH)
Peroxynitrite (ONOO−)

such as cytochrome P450 and nitric oxide synthase. Additionally, ROS can be pro-
duced by the leakage of electrons from the ETC (Fig. 1.1), which is regarded as the
main ROS source in most cells and xanthine oxidase [4, 21, 22].
The univalent reduction of oxygen in the mitochondria generates superoxide
anion, which can be further reduced by the action of mitochondrial superoxide
dismutase into hydrogen peroxide [21]. As previously mentioned, ubisemiqui-
none, a component of mitochondrial respiratory chain, is the most common site
for this univalent reduction of oxygen. While small amounts of reduced oxygen
exist in all functional cells, large quantities of superoxide anion and its derivatives
are generated by neutrophils and activated macrophages via the NADPH oxidase
pathway [4].
Another ROS production pathway involves xanthine oxidase (XO). The conver-
sion of hypoxanthine into xanthine by XO and xanthine into uric acid yields superox-
ide radicals [23]. But only a small amount of ROS is released by the activity of XO
under normal circumstances. However, increased degradation of ATP as a result of
metabolic stress activates XO leading to increased production of superoxide anion
[23]. As a matter of fact, XO has been implicated as a major contributor to ROS levels
in certain pathological conditions involving ischemia and reperfusion [24].
Inflammation and infection also play a significant role in generating ROS.
For instance, a high production of ROS has been noted in endothelial cells
induced by tumor necrosis factor (TNF) [25], lymphocytes can produce ROS via
the 5-­lipoxygenase (5-LO) pathway possibly with CD 40 ligation [26], and cyclo-
oxygenase-­1 generates ROS after stimulated by different chemicals including
TNFα, interleukin-1, bacterial lipopolysaccharide, and tumor promoter
4-O-tetradecanoylphorbol-13-acetate (TPA).
Interestingly, oxidation of dopamine can also lead to ROS production and this
has been shown to be related to neurodegenerative diseases like Parkinson’s
disease [4].

1.1.3.1  Physiological Roles

ROS are formed during normal cellular metabolism. They are involved in many
physiological processes, including the activation of the immune system. For
instance, phagocytes activated as part of the immune system release hydrogen per-
oxide to kill the pathogens. Superoxide anion is also known to have a role in fighting
6 G. Ahmad et al.

Table 1.2  Radical and RNS

non-radical reactive nitrogen Free radicals Non-radicals
species (RNS)
Nitric oxide (•NO−) Nitrogen dioxide: NO2
Nitrous acid (HNO2−) Dinitrogen tetraoxide: N2O4

against infections [1, 27]. On the other hand, ROS released by non-­phagocytic cells,
including fibroblast, endothelial cells and cardiac myocytes is involved in regulating
different intercellular signaling pathways. These cells contain various NADPH oxidase
isoforms [4, 5, 28, 29]. Furthermore, physiological amount of ROS is required in
spermatozoa capacitation, hyper-activation, acrosome reaction and sperm-oocyte
fusion [30].

1.1.4  Reactive Nitrogen Species (RNS)

Reactive Nitrogen species (RNS) are special forms of ROS that contain nitrogen
[2]. Similar to ROS, RNS can also include radicals such as nitric oxide (NO) and
nitrous acid (HNO2) and non-radicals such as nitrogen dioxide (NO2) and dinitro-
gen tetraoxide (N2O4) (Table  1.2). Nitric oxide synthase (NOS) can convert l-­
arginine and oxygen to NO and l-citrulline in the presence of multiple cofactors,
including calcium, NADPH, flavin mononucleotide (FMN), calmodulin and flavin
adenine dinucleotide (FAD) [31]. Furthermore, different forms of RNS including
nitrosonium cation (NO+), nitroxyl anion (NO−) or peroxynitrite (ONOO−) can be
produced as a result of the interactions of NO with various chemicals. Although NO
is not reactive enough to cause DNA damage, NO can react with superoxide anion
to produce peroxynitrite (ONOO−) [32].

1.1.4.1  Physiological Roles

RNS are involved in many physiological mechanisms and processes including

production of several hormones and regulation of inflammatory response by inhibiting
platelets aggregation and preventing the margination of neutrophils to endothelial
cells [33, 34].
NO can alter the heme moiety of guanlate cyclase resulting in alteration in the
structure of the enzyme leading to the activation of the enzyme. The activated form
of guanylate cyclase results in the production of cGMP, which controls the activity
of phosphodiesterases, protein kinases and ion channels. These modulations are
required for many physiological processes including the control of the smooth muscle
tone and suppressing platelet adhesion [35].
1  Overview and Sources of Reactive Oxygen Species (ROS) in the Reproductive System 7

1.1.5  Sources of ROS in Male Reproductive System

1.1.5.1  Generation of ROS

Superoxide anion is considered to be the most abundant ROS in human sperm and it
is the precursor of other ROS types. Accepting another electron to form hydrogen
peroxide and reacts with other radicals such as nitric oxide to form peroxynitrate can
further reduce superoxide anion. Finally, it can also react with itself by dismutation, a
reaction that is catalyzed by SOD to produce hydrogen peroxide. In the presence
of catalase and a transition metal, hydrogen peroxide is converted to the hydroxyl
radical, one of the strongest oxidants in nature [9].
The generation of such ROS occurs via two methods: (1) in the cell membranes,
using NADPH oxidases, and (2) in the mitochondria, using NADH oxido-reductase.

NADPH Oxidases

NADPH oxidase enzymes are located in the cell membranes of the spermatozoa [9].
NADPH oxidases are a group of enzymes that catalyses the conversion of molecular
bivalent oxygen to superoxide. The substrates for these reactions are NADPH
molecules generated by the hexose monophosphate shunt. NOX 5 gene was found
to be expressed in NADPH oxidase in the spermatozoa, located both in the flagella/
neck region and the acromsome and hence, may involve in this process [36].

Electron Leakage in the Mitochondria

ROS are generated by sperm mitochondria under normal cellular respiration.

Spermatozoa are rich in mitochondria because they constantly require ATP for
motility [37]. Electron leakage from cytochromes in the ETC sources the monova-
lent reduction of oxygen to the superoxide anion. There is a link between ROS
production and NADH oxido-reductases activity in the inner and outer mitochon-
drial membranes within respiring spermatozoa. NADH oxido-reductase (ubiqui-
none oxido reductase) catalyses electrons transfer from NADH to coenzyme Q10 in
the ETC. Electron leakage occurs during reverse electron transfer, which acts as a
source of electrons for the reduction of oxygen to the superoxide anion.
Hyperoxic conditions also increase ROS production [38]. When sperm respira-
tion increases due to an increased need for ATP, the concentration of molecular
oxygen in the matrix of the mitochondria increases thus leading to increased
superoxide production as well. However, it is interesting to note that ROS produc-
tion also increases under hypoxic conditions as ROS producing sites in mitochon-
dria have a higher affinity for oxygen than the cytochrome oxidases under such
conditions [39].
8 G. Ahmad et al.

1.1.5.2  Sources of ROS in Seminal Plasma

Immature/Abnormal Spermatozoa

A marked difference has been noted in the quantity of ROS produced by spermato-
zoa at different stage of maturation, suggesting that the level of maturation affects
ROS production [40].
Immature spermatozoa are characterized by retaining excessive residual cyto-
plasm (ERC) around their mid-piece, which is normally expelled during spermio-
genesis. ERC contains high levels of glucose-6-phosphate dehydrogenase (G6PDH),
a cytosolic enzyme that utilizes the hexose monophosphate shunt to produce abnor-
mally high levels of NADPH. Excessive NADPH results in a greater production of
superoxide anions by NADPH oxidases [41]. A positive correlation of high ROS
levels with excessive cytoplasmic retention in spermatozoa is documented [42].
Furthermore, abnormal morphological sperm produce more ROS than their nor-
mal counterparts [43]. In one study, a negative correlation was observed between
the production of ROS and the number of healthy sperm with normal or borderline
morphology. In the same study, a positive correlation of was seen between ROS
production and the number of sperm with amorphous heads, tail defects, excess
cytoplasm and abnormal midpiece [44].

Leukocytes

Activated leukocytes (peroxidase positive) produce large quantities of ROS and it

has been shown that leukocytes are the predominant source of ROS in raw human
semen. ROS produced by leukocytes is believed to account for toxicity against
human sperm.
Peroxidase positive leukocytes are primarily macrophages and polymorphonu-
clear (PMN) leukocytes originating from the prostate and seminal vesicles [40].
Such leukocytes are often found in the seminal fluid. In one study in which seminal
oxidative stress (OS) was artificially-induced, removal of leukocytes by
“Dyanabeads” reduced ROS levels significantly [45].
Although it is generally accepted that an association exists between the number
of leukocytes in the seminal fluid and excessive ROS production, OS can still result
in the absence of leukocytospermia. Of note, one study reported that ROS produc-
tion from PMN up to a concentration of 0.5 × 106/ml is negligible [46].
On the contrary, when PMN leukocytes are activated by stimuli like infection
and/or inflammation, a discharge of ROS ensues [47]. For instance, neutrophils that
contain myeloperoxidase use superoxide anions to oxidize chloride ions. This results
in the production of hypochlorous acid (HOCL), which acts as an anti-­bacterial agent
but is also a kind of ROS. Moreover, leukocytes increase the production of NADPH
by the hexose monophosphate shunt, thus increasing the activity of NADPH oxidases
that ultimately increase the production of superoxide anion.
1  Overview and Sources of Reactive Oxygen Species (ROS) in the Reproductive System 9

As a matter of fact, a strong association exists between the presence of male

accessory gland infections (MAGI) and seminal ROS levels. Prostatitis, prostato-­
vesiculitis and prostato-vesiculo-epididymitis are the commonest MAGI; the most
prevalent being prostatitis. The immune response to MAGI results in an increased
number of leukocytes in the male genital tract, which subsequently leads to ROS
production [9].
Studies have also linked bacterial prostatitis and the autoimmune response to
prostate antigens with excessive ROS production [48]. Although it is generally
accepted that granulocytes in the prostatic fluid are a source of ROS, semen of indi-
viduals suffering from chronic prostatitis that do not contain leukocytes may also
have elevated ROS levels, suggesting that another source of ROS exist in the pros-
tate gland.
The degree of OS is increased when the prostatitis is associated with seminal
vesiculitis. Along the same lines, when compared to prostato-vesiculitis and prosta-
titis, a higher number of white blood cells and ROS levels are observed in prostato-­
vesiculo-­epididymitis [49].

Varicocele

Varicocele is the dilatation of veins in pampiniform plexus around the spermatic

cord. Studies have associated varicocele with many abnormal sperm parameters,
including DNA damage. The latter has been linked to increased levels of ROS and
OS. The higher the varicocele grade, the higher the ROS production [30].

1.2  Conclusion

Despite the strong evidence indicating that animal sperm make ROS, the ability of
human sperm to produce significant amounts of ROS is surrounded by controversy.
Notwithstanding, some evidence that human sperm produce ROS exists but experi-
ments should ensure that sperm preparations are free of leukocytes, since leukocytes
are prolific sources of ROS.

1.2.1  Sources of ROS in Female Reproductive System

While the role of ROS has been extensively investigated in the male reproductive
system, the literature is relatively poor in studies addressing the role of ROS in the
female counterpart. Nonetheless, ROS have been shown to be associated with
pathologies of the female reproductive system, including preeclampsia, polycystic
ovary syndrome, recurrent pregnancy loss and endometriosis [3].
10 G. Ahmad et al.

In the female reproductive system, ROS are mainly found in the cells of ovarian
follicles, the fallopian tube, the endometrium and the peritoneum.

1.2.2  Steroidogenesis and Ovulation

Completion of meiosis I during oocyte maturation is induced by an increase in ROS

and is inhibited by an increase in antioxidants. The contrary occurs during meiosis
II, thus indicating that ROS play an important role during the pre-ovulatory states
[50]. Besides, steroidogenesis is also linked to increased ROS production in the
ovaries by utilizing the cytochrome P450 pathway [51].
An earlier study in the rat suggested that ROS levels in corpus luteum increased
during the regression phase [52]. Newer discoveries shed light to the relationship
between different hormones and ROS production in the luteal phase. In the growing
follicle, ROS induces apoptosis whereas FSH and reduced glutathione (GSH) are
anti-apoptotic. FSH stimulates estrogen production within the granulosa cells,
which in turn increases catalase that inhibits apoptosis [53].
On the contrary, excessive production of LH increases ROS production. The LH
surge in the ovulatory is followed by a post-surge inflammatory phase. If LH secre-
tion is in excess, the post-surge inflammatory precursors may increase thus causing
OS-induced damage due to the overly produced ROS [21, 54].
After ovulation, the corpus luteum produces progesterone. It was found that both
copper/zinc-superoxide dismutase (Cu/Zn-SOD) and progesterone levels increase
in the early-mid luteal phase but decline in the regression phase. Lipid peroxide
opposes Cu/Zn-SOD and progesterone in both phases [21, 54]. On the contrary,
manganese-superoxide dismutase (Mn-SOD) is not affected by progesterone levels
and is independent in its action, whereby its levels are sustained to protect the luteal
cells from OS-induced inflammation [21].

1.2.3  Graffian Follicle

The Graffian follicle has been studied as a potential source of ROS because it con-
tains macrophages, neutrophils, granulosa cells and the oocyte [21].
The oocyte has shown to be a source of ROS by different mechanisms. First,
oxidative phosphorylation within the oocyte utilizes O2 to produce energy (ATP).
Second, the XO system eliminates toxic wastes in the form of uric acid, but at the
expense of generating ROS. Lastly, the NADPH oxidase system whereby the oxida-
tive burst produces HOCl to kill microbes. All these systems are prominent sources
of ROS in the oocyte [21].
Aging is defined by the gradual loss of organ and tissue functions. The ageing of
oocytes was linked with ROS production that may increase oocyte damage [20].
1  Overview and Sources of Reactive Oxygen Species (ROS) in the Reproductive System 11

This hypothesis has led researchers to speculate that ROS might be related to the
occurrence of congenital anomalies in neonates seen originated form older women
[21]. It has been observed that oocytes from women at advanced reproductive age
carry dysfunctions at the mitochondrial DNA level and chromosomal aneuploidy
The marker that has been utilized to assess mitochondrial DNA (mtDNA) damage
is an oxidized derivative of the deoxyguanosine base, namely oxodeoxyguanosine
(8-OHdG) [54].
Menstrual blood is one pivotal way females lose iron (Fe) As discussed earlier,
Fe potentiates oxidative damage—and its build-up in the body poses a higher risk
on menopausal women to OS-related diseases [21]. Menopause also causes
decreased production of estrogen along with its protective properties against oxida-
tive damage of the endometrium [55].

1.2.4  Granulosa Cells and Cumulus Mass Cells

The granulosa cells are one of the main sources of ROS in the graffian follicle.
Undifferentiated granulosa cells give rise to the cumulus oophorus, which anchors
the oocyte and keeps it in place. SOD is the main antioxidant present in these cells
to counteract the ROS production [20, 56]. OS in the granulosa cells and cumulus
oophorus may damage the cell contents, including DNA [54]. There has been shown
that a positive correlation between 8-OHdG levels in the granulosa cells and poor
oocyte quality [57, 58] but the exact mechanisms of ROS production in the granu-
losa cells and cumulus mass remain unclear.

1.2.5  Follicular Fluid

ROS and antioxidants are found in the follicular fluid (FF), which levels have
been linked with oocyte quality [50]. Since FF is produced by both granulosa
cells and theca cells interna [59], it is likely these elements are the sources of
ROS and antioxidants. The most abundant antioxidants in the follicular fluid are
non-enzymatic, including Vitamin C, Vitamin E, glutathione (GSH), hypotaurine
and taurine [50].
The FF has high metabolic activity and contains steroid hormones, growth fac-
tors, leukocytes, cytokines and granulosa cells, all of which are known to increase
ROS production [50].
ROS levels have been studied in the FF of women trying to conceive via
assisted reproductive technology (ART). Total Antioxidant Capacity (TAC) were
lower in those who fail to conceive [60]. In another study, TAC levels were shown
to be higher in FF aspirates that yielded oocytes that successfully fertilized via
ART [61].
12 G. Ahmad et al.

1.2.6  Fallopian Tube

Nitric oxide (NO) synthase is present in human tubal cells, which catalyzes the
formation of NO, the most common source of reactive nitrogen species (RNS). NO is
a potent vasodilator, and its presence in the tubal cells promotes fallopian tube contrac-
tions. These contractions aid to move the oocyte as well as spermatozoa [62]. Increased
NO levels protect the fallopian tubes from microbes, but such levels are also toxic to
spermatozoa. Conversely, decreased NO levels is associated with impaired tube
contractions, with negative effects on both spermatozoa and ovum [54].
As mentioned earlier, NO is formed from O2 and l-arginine, and it further reacts
with superoxide anion to form peroxynitrite. Peroxyinitrite induces lipid peroxidation
and nitration of tyrosine molecules on enzymes essential for signal transduction.
There are essentially three types of NOS: endothelial (eNOS), neuronal (nNOS),
and inducible (iNOS). eNOS has vasodilator effects during pregnancy and its produc-
tion is stimulated by LH and hCG. iNOS is mainly associated with pathological condi-
tions as it is found within macrophages that await stimulation by cytokines [3]. High
levels of NOS over stimulate the tyrosine-dependent enzymes and cause cytotoxicity.
On the other hand, low levels of NOS due to absence of l-arginine is associated with
increased superoxide anion levels, thus causing OS. NO absence has been shown to be
a key factor in disruption of the vascular system that leads to infertile states [54].
Notably, the fallopian tubes are rich in antioxidant enzymes [3, 20, 21, 50, 54,
63–69]. The effect of ROS on sperm capacitation can be blocked by these antioxi-
dants. Alternative sources of physiological ROS, possibly generated by the cumulus
cells might promote capacitation and prepare the sperm to fertilize.

1.2.7  Peritoneal Cavity

The main sources of ROS in the peritoneal cavity are macrophages, endometrial
cells, red blood cells and menstrual-reflux debris [65]. Idiopathic infertile women
were shown to have elevated levels of peritoneal leukocytes, up to 95 % of which are
macrophages. Increased levels of malonaldehyde, a stable end-product of lipid
peroxidation, was found in patients with idiopathic female infertility [21, 39, 65, 66,
70, 71]. Furthermore, the presence of higher level of ROS and lower level of anti-
oxidants in the peritoneum has been suggested to cause endometriosis, a common
cause of female infertility [63].

1.2.8  Endometrium

ROS levels in the physiological range generated within endometrial cells ultimately
increase in prostaglandin F 2alpha (PGF2α), which in turn plays a role in luteal
regression [72]. Moreover, withdrawal of estrogen and progesterone causes a
1  Overview and Sources of Reactive Oxygen Species (ROS) in the Reproductive System 13

decrease in SOD levels and a subsequent increase in ROS levels, which promotes
NF-KB transcription factor to express cyclo-oxygenase-2 (COX-2) enzyme. COX-2
will catalyze the synthesis of PGF2a that contributes to the process of endometrial
shedding [73].

1.3  Conclusion

Although there is strong evidence indicating that ROS are generated by several cel-
lular elements and systems in the female reproductive tract, the role of ROS remains
to be further elucidated.

1.3.1  Oxidative Stress (OS)

As highly reactive molecules, ROS readily react with a wide range of biological
molecules, including unsaturated fatty acids, sulphydryl proteins and nucleic acids
[8]. In combating the ROS, there is antioxidant defense mechanism in our body,
which are enzymes or non-enzymes that are able to neutralize these reactive mole-
cules. The loss of the required balance between the production of ROS and the
elimination of these radicals by the antioxidant defense system leads to a condition
known as oxidative stress (OS). This can be caused by either high ROS production
or decreased antioxidants or even both of them together. As discussed earlier, ROS
are formed during the normal cellular metabolism. However, when ROS levels
increase beyond the normal capacity of antioxidant defense, OS occurs and can
result in cellular dysfunction or cell death through different mechanisms including
lipid peroxidation, nucleic acid and protein damages directly or indirectly [8].
Apoptosis is defined as a unique form of programmed cell death, which plays a
significant role in controlling the growth and homeostasis of different organisms
[4]. An association between increased ROS levels and apoptosis is also well docu-
mented [74, 75].
The extent of these damages due to OS depends on several factors, which include
the extent of anti-oxidation in the environment and lipid saturation. As unsaturated
fatty acids contain unconjugated double bonds adjacent to the methylene groups,
this makes the methylene carbon-hydrogen bonds weaker and thus more susceptible
to peroxidative damage [76]. Other factors like the presence of metal binding pro-
teins (MBP), including transferrin and albumin [77, 78] also seem to play a role;
these molecules help to bind iron that are present in the cell, which would otherwise
potentiate the oxidative damage.
Due to its damaging effect on the cells, OS is related to many pathological
conditions and diseases including cancer, ischemia and infertility, as well as non-­
pathological process including aging [3]. The negative effects of OS to the male and
female reproductive systems can be found in Chap. 4.
14 G. Ahmad et al.

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